A major dose limiting toxicity of chemoradiotherapy of non-small cell carcinoma of the lung is irradiation esophagitis. This application seeks to prove that ionizing irradiation limits reparative stem cell engraftment of the irradiated esophagus through continuous reactive oxygen species (ROS) production by cells of the microenvironment, and that the mechanism of amelioration of toxicity by intraesophageal MnSOD-Plasmid Liposome (PL) (antioxidant) gene therapy is by stabilizing the antioxidant pool. We have demonstrated that intra-esophageal administration of MnSOD-PL decreases the toxicity of single fraction or fractionated radiotherapy to the mouse esophagus, decreases irradiation-induced glutathione and thiol depletion, lipid peroxidation, and enhance the survival of intravenously injected esophageal stem cells isolated by either side population (SP) cell sorting or 7-day pre-plate technique. We propose to utilize MnSOD-PL antioxidant therapy to enhance engraftment by reducing ROS production by the microenvironment. We will use three assays for measuring irradiation-induced toxic ROS effects on bone marrow derived esophageal stem cell engraftment: 1) clonal homologous recombination (HR) in donor male C57BL/6J (FYDR) cells;2) fusion of donor cells with recipient esophagus;and 3) apoptosis of both resident and engrafted esophageal stem cells. The first specific aim tests the hypothesis that homing and proliferation of bone marrow origin serially transplantable esophageal progenitor cells from G418r LacZ+ (B6.129S7-Gt(ROSA)26Sor/J) male mice to the irradiated female recipient esophagus results in cell dose dependent irradiation protection, and correlates with recovery of G418r LacZ+ colony forming squamous cell progenitors in vitro. The second specific aim tests the hypothesis that irradiation-induced ROS, specifically peroxynitrite, in the microenvironment of the irradiated female mouse esophagus induces HR in donor C57BL/6J male (FYDR) marrow SP cells quantitated as clonal yellow foci, cell fusion with recipient esophageal cells and apoptosis. The third specific aim tests the hypothesis that restoration of antioxidant reserves in the irradiated esophagus by MnSOD-PL treatment, in both ROSA and FYDR marrow cell transplantation models, stabilizes marrow stem cell engraftment. Methods include engraftment of donor esophageal and hematopoietic stem cell subsets from male C57BL/6J, FYDR, and selectable G418 resistant LacZ (ROSA) male donors into irradiated recipient C57BL/6J female GFP+ mice, cell sorting, FISH, biochemical measurements of antioxidants in vitro and in vivo, and assays for direct and bystander killing. These studies should elucidate the molecular mechanism of induction of damage to engrafting marrow origin, epithelial stem cells by the irradiated esophageal microenvironment and identify new targets for therapeutic intervention.